EFFICIENT AND SPECIFIC AMPLIFICATION OF IDENTIFIED PARTIAL DUPLICATIONS OF HUMAN MITOCHONDRIAL-DNA BY LONG PCR

The use of PCR to identify mtDNAs containing a partial duplication (du p-mtDNA) in the presence of a heteroplasmic population of mtDNAs harbo ring the corresponding deletion (Delta-mtDNA) leads to ambiguous resul ts: when the primers anneal in the duplicated portion of the dup-mtDNA (which is also the non-deleted region of the Delta-mtDNA) and point t owards the abnormal breakpoint junction, both templates are amplified indiscriminately. We have developed two different 'long PCR' approache s to amplify dup-mtDNA even in the presence of Delta-mtDNA and wild-ty pe mtDNA (wt-mtDNA). Long PCR with two primers annealing in the non-du plicated region in dup-mtDNA (equivalent to the region missing in Delt a-mtDNA) and whose 3' ends pointed towards the duplicated area amplifi ed both dup-mtDNA and coexisting wt-mtDNA. We observed, however, a pre ferential amplification of the wt-mtDNA over that of the longer dup-mt DNAs. This problem was partly overcome by modifying the PCR conditions (extension time, amplicon length, amount of template). In order to ov ercome the problem of co-amplification, we developed a novel PCR metho d to amplify specifically dup-mtDNAs. A forward primer annealing acros s the breakpoint junction was used in conjunction with a backward prim er annealing in the non-duplicated region. For those duplication break points flanked by direct repeats, we designed a 'breakpoint loop-out' primer whose sequence omitted the repeated region, in order to avoid t he annealing of this primer to wt-mtDNA. This second approach was able to amplify specifically and efficiently the dup-mtDNA in all samples analyzed, irrespective of the size of the duplication or its proportio n in the samples.